LOCUS       Exported                5863 bp ds-DNA     circular SYN 08-MAR-2016
DEFINITION  Gateway Entry vector encoding one TALEN (right) of a pair targeting 
            the AAVS1 locus within intron 1 of the human PPP1R12C gene.
ACCESSION   .
VERSION     .
KEYWORDS    KW227_pTALdNC-AAVS1_T2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5863)
  AUTHORS   Oceguera-Yanez F, Kim SI, Matsumoto T, Tan GW, Xiang L, Hatani T, 
            Kondo T, Ikeya M, Yoshida Y, Inoue H, Woltjen K.
  TITLE     Engineering the AAVS1 locus for consistent and scalable transgene 
            expression in human iPSCs and their differentiated derivatives.
  JOURNAL   Methods 2015.
  PUBMED    26707206
REFERENCE   2  (bases 1 to 5863)
  AUTHORS   Knut Woltjen
  TITLE     Direct Submission
  JOURNAL   Exported Sunday, Jul 3, 2016 from SnapGene 3.1.4
            http://www.snapgene.com
COMMENT     ID: RDB13731
            Relation: Use as a pair in combination with expression vector 
            containing KW226 pTALdNC-AAVS1_T1 (RDB13730)
            Remarks: Gateway Entry vector. Requires cloning into an expression 
            vector before use. Also compatible with standard 
            restriction/ligation (SalI/XhoI or EcoRV).
FEATURES             Location/Qualifiers
     source          1..5863
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          join(1020..1045,1985..2043)
                     /organism="pFUS_A30A"
                     /mol_type="other DNA"
                     /label=source:pFUS_A30A
                     /note="source:pFUS_A30A"
                     /note="Cermak et al., Nucleic Acids Research 2011, 
                     PMID:21493687"
     source          1050..1119
                     /organism="pNN1"
                     /mol_type="other DNA"
                     /label=source:pNN1
                     /note="source:pNN1"
     source          1124..1217
                     /organism="pNN2"
                     /mol_type="other DNA"
                     /label=source:pNN2
                     /note="source:pNN2"
     source          1222..1316
                     /organism="pNN3"
                     /mol_type="other DNA"
                     /label=source:pNN3
                     /note="source:pNN3"
     source          1321..1402
                     /organism="pNN4"
                     /mol_type="other DNA"
                     /label=source:pNN4
                     /note="source:pNN4"
     source          1407..1495
                     /organism="pHD5"
                     /mol_type="other DNA"
                     /label=source:pHD5
                     /note="source:pHD5"
     source          1500..1594
                     /organism="pNG6"
                     /mol_type="other DNA"
                     /label=source:pNG6
                     /note="source:pNG6"
     source          1599..1692
                     /organism="pNG7"
                     /mol_type="other DNA"
                     /label=source:pNG7
                     /note="source:pNG7"
     source          1697..1792
                     /organism="pNG8"
                     /mol_type="other DNA"
                     /label=source:pNG8
                     /note="source:pNG8"
     source          1797..1892
                     /organism="pNG9"
                     /mol_type="other DNA"
                     /label=source:pNG9
                     /note="source:pNG9"
     source          1897..1980
                     /organism="pHD10"
                     /mol_type="other DNA"
                     /label=source:pHD10
                     /note="source:pHD10"
     source          join(2048..2065,3005..3059)
                     /organism="pFUS_A30B"
                     /mol_type="other DNA"
                     /label=source:pFUS_A30B
                     /note="source:pFUS_A30B"
                     /note="Cermak et al., Nucleic Acids Research 2011, 
                     PMID:21493687"
     source          2070..2139
                     /organism="pNG1"
                     /mol_type="other DNA"
                     /label=source:pNG1
                     /note="source:pNG1"
     source          2144..2237
                     /organism="pNN2"
                     /mol_type="other DNA"
                     /label=source:pNN2(1)
                     /note="source:pNN2(1)"
     source          2242..2336
                     /organism="pNG3"
                     /mol_type="other DNA"
                     /label=source:pNG3
                     /note="source:pNG3"
     source          2341..2422
                     /organism="pHD4"
                     /mol_type="other DNA"
                     /label=source:pHD4
                     /note="source:pHD4"
     source          2427..2515
                     /organism="pNI5"
                     /mol_type="other DNA"
                     /label=source:pNI5
                     /note="source:pNI5"
     source          2520..2614
                     /organism="pHD6"
                     /mol_type="other DNA"
                     /label=source:pHD6
                     /note="source:pHD6"
     source          2619..2712
                     /organism="pHD7"
                     /mol_type="other DNA"
                     /label=source:pHD7
                     /note="source:pHD7"
     source          2717..2812
                     /organism="pNI8"
                     /mol_type="other DNA"
                     /label=source:pNI8
                     /note="source:pNI8"
     source          2817..2912
                     /organism="pNI9"
                     /mol_type="other DNA"
                     /label=source:pNI9
                     /note="source:pNI9"
     source          2917..3000
                     /organism="pNG10"
                     /mol_type="other DNA"
                     /label=source:pNG10
                     /note="source:pNG10"
     source          join(3064..3085,3262..3289)
                     /organism="pFUS_B2"
                     /mol_type="other DNA"
                     /label=source:pFUS_B2
                     /note="source:pFUS_B2"
                     /note="Cermak et al., Nucleic Acids Research 2011, 
                     PMID:21493687"
     source          3090..3159
                     /organism="pHD1"
                     /mol_type="other DNA"
                     /label=source:pHD1
                     /note="source:pHD1"
     source          3164..3257
                     /organism="pHD2"
                     /mol_type="other DNA"
                     /label=source:pHD2
                     /note="source:pHD2"
     source          3294..3378
                     /organism="pLR-NG"
                     /mol_type="other DNA"
                     /label=source:pLR-NG
                     /note="source:pLR-NG"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /note="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(106..149)
                     /label=rrnB T1 txn terminator
                     /note="rrnB T1 txn terminator"
     terminator      complement(281..308)
                     /label=rrnB T2 txn terminator
                     /note="rrnB T2 txn terminator"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attL1"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             490..1015
                     /codon_start=1
                     /label=TAL-N'del5'
                     /note="TAL-N'del"
                     /note="truncated TALE architecture for increased nuclease 
                     activity
                     Sakuma et al., Genes Cells 2013 PMID:23388034"
                     /translation="MASSPPKKKRKVSWKDASGWSRMHAAPRRRAAQPSDASPAAQVDL
                     RTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVTYQHII
                     TALPEATHEDIVGVGKQWSGARALEALLTDAGELRGPPLQLDTGQLVKIAKRGGVTAME
                     AVHASRNALTGA"
     misc_signal     505..525
                     /note="SV40 NLS"
     CDS             526..552
                     /product="epitope tag from the baculovirus Autographa 
                     californica GP64 envelope fusion protein"
                     /note="AcV5 tag"
     misc_feature    1016..1019
                     /label=Esp3I-OH #1
                     /note="Esp3I-OH #1"
     misc_feature    1046..1049
                     /label=BsaI-OH #1
                     /note="BsaI-OH #1"
     misc_feature    1120..1123
                     /label=BsaI-OH #2
                     /note="BsaI-OH #2"
     misc_feature    1218..1221
                     /label=BsaI-OH #3(1)
                     /note="BsaI-OH #3(1)"
     misc_feature    1317..1320
                     /label=BsaI-OH #4
                     /note="BsaI-OH #4"
     misc_feature    1403..1406
                     /label=BsaI-OH #5
                     /note="BsaI-OH #5"
     misc_feature    1496..1499
                     /label=BsaI-OH #6(1)
                     /note="BsaI-OH #6(1)"
     misc_feature    1595..1598
                     /label=BsaI-OH #7
                     /note="BsaI-OH #7"
     misc_feature    1693..1696
                     /label=BsaI-OH #8
                     /note="BsaI-OH #8"
     misc_feature    1793..1796
                     /label=BsaI-OH #9(1)
                     /note="BsaI-OH #9(1)"
     misc_feature    1893..1896
                     /label=BsaI-OH #10
                     /note="BsaI-OH #10"
     misc_feature    1981..1984
                     /label=BsaI-OH #11(1)
                     /note="BsaI-OH #11(1)"
     misc_feature    complement(2044..2047)
                     /label=Esp3I-OH #1a
                     /note="Esp3I-OH #1a"
     misc_feature    2066..2069
                     /label=BsaI-OH #1(2)
                     /note="BsaI-OH #1(2)"
     misc_feature    2140..2143
                     /label=BsaI-OH #2(2)
                     /note="BsaI-OH #2(2)"
     misc_feature    2238..2241
                     /label=BsaI-OH #3(2)
                     /note="BsaI-OH #3(2)"
     misc_feature    2337..2340
                     /label=BsaI-OH #4(1)
                     /note="BsaI-OH #4(1)"
     misc_feature    2423..2426
                     /label=BsaI-OH #5(1)
                     /note="BsaI-OH #5(1)"
     misc_feature    2516..2519
                     /label=BsaI-OH #6
                     /note="BsaI-OH #6"
     misc_feature    2615..2618
                     /label=BsaI-OH #7(1)
                     /note="BsaI-OH #7(1)"
     misc_feature    2713..2716
                     /label=BsaI-OH #8(1)
                     /note="BsaI-OH #8(1)"
     misc_feature    2813..2816
                     /label=BsaI-OH #9
                     /note="BsaI-OH #9"
     misc_feature    2913..2916
                     /label=BsaI-OH #10(1)
                     /note="BsaI-OH #10(1)"
     misc_feature    3001..3004
                     /label=BsaI-OH #11
                     /note="BsaI-OH #11"
     misc_feature    3060..3063
                     /label=Esp3I-OH #2
                     /note="Esp3I-OH #2"
     misc_feature    3086..3089
                     /label=BsaI-OH #1(1)
                     /note="BsaI-OH #1(1)"
     misc_feature    3160..3163
                     /label=BsaI-OH #2(1)
                     /note="BsaI-OH #2(1)"
     misc_feature    3258..3261
                     /label=BsaI-OH #3
                     /note="BsaI-OH #3"
     misc_feature    3290..3293
                     /label=Esp3I-OH #3
                     /note="Esp3I-OH #3"
     misc_feature    3379..3382
                     /label=Esp3I-OH #4
                     /note="Esp3I-OH #4"
     CDS             3383..3468
                     /codon_start=3
                     /label=TAL-C'(nls)del5' + FokI
                     /note="TAL-C'del"
                     /note="truncated TALE architecture for increased nuclease 
                     activity
                     Sakuma et al., Genes Cells 2013 PMID:23388034"
                     /translation="HLVALACLGGRPAMDAVKKGLPHAPELI"
     CDS             3475..4062
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /note="FokI cleavage domain"
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     protein_bind    complement(4092..4191)
                     /gene="mutant version of attL"
                     /bound_moiety="LR Clonase(TM)"
                     /note="attL2"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4314..5123
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR (delEsp3I)"
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      5213..5801
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacacattga tgagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac
      481 tggatcacca tggcttcctc ccctccaaag aaaaagagaa aggttagttg gaaggacgca
      541 agtggttggt ctagaatgca tgcggccccg cgacggcgtg ctgcgcaacc ctccgacgct
      601 tcgccggccg cgcaggtgga tctacgcacg ctcggctaca gtcagcagca gcaagagaag
      661 atcaaaccga aggtgcgttc gacagtggcg cagcaccacg aggcactggt gggccatggg
      721 tttacacacg cgcacatcgt tgcgctcagc caacacccgg cagcgttagg gaccgtcgct
      781 gtcacgtatc agcacataat cacggcgttg ccagaggcga cacacgaaga catcgttggc
      841 gtcggcaaac agtggtccgg cgcacgcgcc ctggaggcct tgctcacgga tgcgggggag
      901 ttgagaggtc cgccgttaca gttggacaca ggccaacttg tgaagattgc aaaacgtggc
      961 ggcgtgaccg caatggaggc agtgcatgca tcgcgcaatg cactgacggg tgcccccctg
     1021 aacctgaccc cggaccaagt ggtggctatc gccagcaaca atggcggcaa gcaagcgctc
     1081 gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa
     1141 gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg
     1201 ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc
     1261 aacaatggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag
     1321 gaccatggcc tgaccccgga ccaagtggtg gctatcgcca gcaacaatgg cggcaagcaa
     1381 gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgactccg
     1441 gaccaagtgg tggctatcgc cagccacgat ggcggcaagc aagcgctcga aacggtgcag
     1501 cggctgttgc cggtgctgtg ccaggaccat ggcctgaccc cggaccaagt ggtggctatc
     1561 gccagcaacg gtggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg
     1621 tgccaggacc atggcctgac cccggaccaa gtggtggcta tcgccagcaa cggtggcggc
     1681 aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg
     1741 accccggacc aagtggtggc tatcgccagc aacggtggcg gcaagcaagc gctcgaaacg
     1801 gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg
     1861 gctatcgcca gcaacggtgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg
     1921 gtgctgtgcc aggaccatgg cctgactccg gaccaagtgg tggctatcgc cagccacgat
     1981 ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat
     2041 ggcctgaccc cggaccaagt ggtggctatc gccagcaacg gtggcggcaa gcaagcgctc
     2101 gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac cccggaccaa
     2161 gtggtggcta tcgccagcaa caatggcggc aagcaagcgc tcgaaacggt gcagcggctg
     2221 ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc
     2281 aacggtggcg gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag
     2341 gaccatggcc tgactccgga ccaagtggtg gctatcgcca gccacgatgg cggcaagcaa
     2401 gcgctcgaaa cggtgcagcg gctgttgccg gtgctgtgcc aggaccatgg cctgaccccg
     2461 gaccaagtgg tggctatcgc cagcaacatt ggcggcaagc aagcgctcga aacggtgcag
     2521 cggctgttgc cggtgctgtg ccaggaccat ggcctgactc cggaccaagt ggtggctatc
     2581 gccagccacg atggcggcaa gcaagcgctc gaaacggtgc agcggctgtt gccggtgctg
     2641 tgccaggacc atggcctgac tccggaccaa gtggtggcta tcgccagcca cgatggcggc
     2701 aagcaagcgc tcgaaacggt gcagcggctg ttgccggtgc tgtgccagga ccatggcctg
     2761 accccggacc aagtggtggc tatcgccagc aacattggcg gcaagcaagc gctcgaaacg
     2821 gtgcagcggc tgttgccggt gctgtgccag gaccatggcc tgaccccgga ccaagtggtg
     2881 gctatcgcca gcaacattgg cggcaagcaa gcgctcgaaa cggtgcagcg gctgttgccg
     2941 gtgctgtgcc aggaccatgg cctgaccccg gaccaagtgg tggctatcgc cagcaacggt
     3001 ggcggcaagc aagcgctcga aacggtgcag cggctgttgc cggtgctgtg ccaggaccat
     3061 ggcctgaccc cggaccaagt ggtggctatc gccagccacg atggcggcaa gcaagcgctc
     3121 gaaacggtgc agcggctgtt gccggtgctg tgccaggacc atggcctgac tccggaccaa
     3181 gtggtggcta tcgccagcca cgatggcggc aagcaagcgc tcgaaacggt gcagcggctg
     3241 ttgccggtgc tgtgccagga ccatggcctg accccggacc aagtggtggc tatcgccagc
     3301 aacggtggcg gcaagcaagc gctcgaaagc attgtggccc agctgagccg gcctgatccg
     3361 gcgttggccg cgttgaccaa cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct
     3421 gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag atcccagcta
     3481 gtgaaatctg aattggaaga gaagaaatct gaacttagac ataaattgaa atatgtgcca
     3541 catgaatata ttgaattgat tgaaatcgca agaaattcaa ctcaggatag aatccttgaa
     3601 atgaaggtga tggagttctt tatgaaggtt tatggttatc gtggtaaaca tttgggtgga
     3661 tcaaggaaac cagacggagc aatttatact gtcggatctc ctattgatta cggtgtgatc
     3721 gttgatacta aggcatattc aggaggttat aatcttccaa ttggtcaagc agatgaaatg
     3781 caaagatatg tcgaagagaa tcaaacaaga aacaagcata tcaaccctaa tgaatggtgg
     3841 aaagtctatc catcttcagt aacagaattt aagttcttgt ttgtgagtgg tcatttcaaa
     3901 ggaaactaca aagctcagct tacaagattg aatcatatca ctaattgtaa tggagctgtt
     3961 cttagtgtag aagagctttt gattggtgga gaaatgatta aagctggtac attgacactt
     4021 gaggaagtga gaaggaaatt taataacggt gagataaact tttaataggc cgccgcactc
     4081 gagatatcta gacccagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc
     4141 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
     4201 tgcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
     4261 catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     4321 atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg
     4381 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg
     4441 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     4501 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     4561 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa
     4621 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     4681 cagtgtccct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     4741 gcgtatttcg tctggctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     4801 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     4861 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     4921 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     4981 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     5041 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     5101 tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacat tattcagatt
     5161 gggccccgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5221 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5281 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5341 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5401 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5461 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5521 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5581 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5641 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5701 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5761 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5821 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtt
//
